Device and method for the detection of particles

ABSTRACT

The present invention relates to devices and methods for the qualitative and/or quantitative detection of particles. In particular, the invention relates to devices for the detection of particles, comprising a reaction chamber formed within a chamber body between a first surface and a second surface, wherein the second surface is located opposite to the first surface, and one or more displacers, wherein the distance between the first surface and the second surface is variable via the one or more displacers at least in one or more parts of the surface area of the first surface and/or second surface. The invention also relates to corresponding methods for the detection of particles.

CLAIM OF PRIORITY

This application claims priority to and is a continuation of applicationSer. No. 12/092,422, filed May 2, 2008, which claims priority under 35USC §371 to International Application No. PCT/EP2006/068153, filed onNov. 6, 2006, which claims priority to German Application Serial No. 102005 052 752.3, filed Nov. 4, 2005, each of which is incorporated byreference in its entirety.

RELATED APPLICATIONS

This application claims the benefit of German patent application DE 102005 052 752, which is incorporated herein by reference in its entirety.The present application further relates to German patent application DE10 2005 052 713 and to the International Patent Application entitled“Method and device for the detection of molecular interactions”, filedon Nov. 6, 2006 (Maiwald reference number: C 7794), both of whichapplications are incorporated herein by reference in their entireties.

FIELD OF THE INVENTION

The invention relates to devices and methods for the detection ofparticles.

BACKGROUND

The detection of the presence and/or the enumeration of absolute levelsof one or more species of particles like cells or viruses in samplessuch as human and non-human body fluids is of primary importance indetermining the state of health of human beings and mammals in general.Clinically important examples of such applications involve counting ofCD 4+ cells in 1-HV-positive subjects, of granulocytes and platelets inpatients treated with chemotherapy, and of leukocytes in blood bags.Non-medical applications, on the other hand, include the detection, of(bacterial) contaminants in environmental samples such as sewage or infood products.

The main analytical platform for performing such analyses is currentlyflow cytometry-based assay systems. Flow cytometry involves the deliveryof a flowing stream containing a sample having target particles thereinto the detection region of a flow cytometer. The particles are arrangedin single file along a core stream using hydrodynamic focusing within asheath fluid. The particles are then individually interrogated by alight beam. Typically, the target particles in the detection region areirradiated using a laser to create an illumination phenomenon by thetarget particles. The optics and detection electronics measure the lightabsorption, scattering, fluorescence, and/or spectral properties of thetarget particles in the sample, or alternatively, the respectiveproperties of a fluorescent label attached to the target particles. Incase of fluorescence, each target particle produces a burst offluorescence photons as it passes through the illumination region.Furthermore, differentiation of the fluorescence from the illuminationor the excitation light can be accomplished with a filter or acombination of filters. Detection of the fluorescence is achieved usinga photomultiplier tube or a photodiode. Another technique relies onlight scattering of photons in the illumination beam by the targetparticle. The target particle is identified by its light scattering as afunction of the angle of scattering, which is, in turn, a function ofits size and shape as well as the wavelength of the scattered photons.

Thus, the successful detection and identification of a single targetparticle depends upon several factors. First, the laser power must besufficient to generate a large enough number of fluorescence (oralternatively scattering) photons during the brief passage of the targetparticle through the irradiation region. Detection of the particletypically occurs when the a sufficient number of photons is generated sothat the fluorescent burst from the target particle is reliablydifferentiated from random fluctuations of background photons. Second,it is important to minimize these unwanted background photons arisingfrom scattering or from fluorescence emitted by the carrier liquid ofthe sample or impurities in the liquid, as well as from the apparatusitself, such as the walls of the capillary through which the flow streampasses.

Flow cytometers and methods for their use in different applications aredescribed, for example, in Sharpiro, R. M. (2002) Practical FlowCytometry, 4th ed., Wiley-Liss, New York, N.Y.; as well as inter alia inWO 90/13368, WO 99/44037, and WO 01/59429.

However, conventional flow cytometry systems remain largely inaccessiblefor routine clinical use due to typically bulky instrumentation, whichdoes not only malce “on-site” measurements (e.g. bedside testing)difficult but also gives rise to high costs per analysis. Thus, there isa clear need for simpler, more compact and less expensive systems,preferably exhibiting comparable performance characteristics.

Accordingly, in recent years microfluidic techniques have been employedfor the purposes of developing cytometers which require smaller sampleand reagent volumes (see, e.g., Altendorf, E. et al. (1997) Sens.Actual. 1, 531-534; Huh, D. et al. (2005) Physiol. Meas. 26, R73-R98;Dittrich, P. S., and Manz, A. (2005) Anal. Bioanal. Chem. 382,1771-1782). Analytical instruments based on these efforts are smallerand more portable than conventional devices.

An example of such a miniaturized flow cytometer is described in theInternational Patent Application WO 02/10713. This device uses anon-precision fluid driver that is coupled to the sample fluid receiverand the reservoirs for supporting fluids, respectively, and controlledby a closed loop feedback path, thus enabling a more compactinstrumental setup.

Instead of using flow cytrometry it is also possible to determine thepresence and/or number of particles in a given sample in an indirectmanner by employing molecular markers (i.e. labels) that are specificfor the particles of interest, and whose copy number in the samplecorrelates with that of the particles. Currently, different approachesare available to perform such analyses, for example ELISA-based assays(see, e.g., Kannangal, R. et al. (2001) Clin. Diagn. Lab. Immunol. 8,1286-1288) as well as microscope-based methods involving the use ofcoated paramagnetic beads (see, e.g., Carella, A. Y. et al. (1995) Clin.Diagn. Lab. Immunol. 2, 623-625) or coated latex beads (see, e.g.,Balakrishnan, P. et al (2004) J. Acquir. Immune Defic. Syndr. 36,1006-1010). Furthermore, it is also possible to specifically capture thelabeled particles on a membrane before imaging them using microscopeoptics (Rodriguez, W. R. et al. (2005) PLOS Medicine 2, el82, 663-672).

Another type of assay devices for counting particles is described in theInternational Patent Application WO 2005/008226. The device comprises alight source projecting light into a sub-area of a sample chipcontaining the particles to be analyzed labeled with a dye, and ashifter for shifting the position of the chip by a predetermineddistance at every predetermined time interval relative to the objectlens and the light source, respectively, in such a way that a certainarea adjacent to the area photographed just before is shifted to thepoint where the light is incident. Therefore, sub-areas on the samplechip are photographed successively. The number of particles in eachsub-area is counted and mathematically processed to calculate the totalnumber of particles in the sample.

Furthermore, the U.S. Patent Application 2006/0024756 relates to acompact imaging cytometry device for the detection of magneticallylabeled target particles or cells. For that purpose, all cells presentin a biological sample to be analyzed are fluorescently labeled, butonly the target cells are also magnetically labeled using a monoclonalantibody coupled to ferromagnetic beads. The labeled sample, in achamber or cuvette, is placed between two wedge-shaped magnets toselectively move the magnetically labeled cells to the observationsurface of the cuvette. An LED illuminates the cells and a CCD cameracaptures the images of the fluorescent light emitted by the targetcells. Digital image analysis provides a count of the cells on thesurface that can be related to the target cell concentration of theoriginal sample.

However, all these devices and methods described above requirecomparably sophisticated detection techniques, which are expensive bothin terms of initial cost and maintenance of the necessary analyticalinstrumentation as well as require highly trained personnel. This makesthe conventional systems unsuitable for routine medical practices,“bedside” testing, or in remote locations.

Thus, there still remains a need for assay devices for the qualitativeand/or quantitative detection of one or more particles in a sample,which overcome the above-mentioned limitations. in particular, there isa need for devices enabling the detection even of small amounts (i.e.numbers) of a given particle not only with high sensitivity but also inan easy-to-do and cost-efficient manner.

Furthermore, there is also a need for corresponding methods using suchassay devices for the rapid and reliable detection of the presenceand/or the accurate determination of the amount of one or more speciesof particles in a given sample. In particular, there is a need formethods that can be performed “on-site”, i.e. during or immediatelyafter collecting the sample to be analyzed.

Accordingly, it is an object of the present invention to provide suchassay devices as well as the corresponding methods using the same.

SUMMARY OF THE INVENTION

In a first aspect, the present invention relates to a device for thequalitative and/or quantitative detection of particles, comprising areaction chamber formed within a chamber body between a first surfaceand a second surface, wherein the second surface is located opposite tothe first surface; and one or more displacers, wherein the distancebetween the first surface and the second surface is variable via the oneor more displacers at least in one or more parts of the surface area ofthe first surface and/or the second surface.

The one or more displacers may be integral parts of the first surfaceand/or of the second surface (“displacement structures”) or may beself-contained entities (“displacement bodies”) located opposite to thefirst surface and/or of the second surface. The one or more displacersmay be made of an elastically deformable material. in a preferredembodiment, the device comprises two displacers, which may be locatedopposite either to the same or to different surfaces.

Preferably, at least a part of the first and/or the second surfaceis/are made of a transparent material. Furthermore, it is preferred thatat least one or more parts of the first surface and/or of the secondsurface is/are elastically deformable. Particularly preferably, the atleast one or more elastically deformable parts are located opposite tothe one or more displacers. The at least one or more elasticallydeformable parts may be different from the surface area where detectiontakes place.

In some embodiments, the device of the invention further comprises oneor more means, which, when the reaction chamber is elastically deformed,allow keeping the volume of the reaction chamber essentially constant.Preferably, the one or more means are elastic sidewalls laterallydelimiting the reaction chamber.

In a further aspect, the present invention relates to a system,comprising at least two parts, wherein each of the at least two partscomprises a reaction chamber formed within a chamber body between afirst surface and a second surface, wherein the second surface islocated opposite to the first surface; and one or more displaeers,wherein the distance between the first surface and the second surface isvariable via the one or more displacers at least in one or more parts ofthe surface area of the first surface and/or the second surface; andwherein the reaction chambers of the at least two parts are incommunication with each other.

The reaction chamber, the first surface, the second surface and/or theat least one displacer of a part of the system may be formed asdescribed for the inventive device.

In one embodiment, the system further comprises one or more means,which, when the reaction chamber is elastically deformed, allow keepingthe volume of the reaction chamber essentially constant. Preferably, theone or more means are elastic sidewalls laterally delimiting thereaction chamber.

In another embodiment, the system further comprises a detection system.

Particularly preferably, the system further comprises a sampleintroduction passage in communication with each of the reaction chambersof the at least two devices and optionally also one or more means thatallow for a transient fluid communication between the at least tworeaction chambers.

In a further aspect, the present invention relates to a method for thequalitative and/or quantitative detection of particles, comprisingpositioning a sample supposed to comprise one or more species ofparticles to be detected in a reaction chamber, displacing at least apart of the sample within the reaction chamber via the one or moredisplacers, and detecting/determining a value indicative for thepresence and/or number or one or more species of particles.

Preferably, the sample to be analyzed is a biological sample, and theone or more species of particles to be detected are selected from thegroup consisting of prokaryotic cells, eukaryotic cells, and viralparticles.

In a preferred embodiment, positioning the sample in a reaction chambercomprises introducing said sample into the reaction chamber of a deviceaccording to the present invention.

Preferably, the at least part of the sample is displaced by varying,particularly preferably by reducing, the distance between the firstsurface and the second surface at least in one or more parts of thesurface area of the first surface and/or the second surface by applyingpressure to the first surface and/or the second surface via at least oneof the one or more displacers.

In a preferred embodiment of the inventive method, after displacing atleast part of the sample the reduced distance is subsequentlyre-increased. This reduction and subsequent re-increase of the distancemay be performed repeatedly. Detection is preferably performed after thedistance between the first surface and the second surface has beenreduced.

In one embodiment, the method of the invention comprises positioning asample comprising multiple particles in a reaction chamber, displacing asubset of said multiple particles within the reaction chamber via theone or more displacers, determining one or more values indicative forthe number of the subset of particles displaced within the reactionchamber, and optionally calculating the total number of the multipleparticles in the reaction chamber from the one or more values obtainedduring detection.

In some embodiments, the method further comprisespositioning/introducing one or more agents each comprising one or moredetectable moieties into the reaction chamber before performingdetection. Preferably, the one or more agents are selected from thegroup consisting of nucleic acids, peptides, protein domains, proteins,carbohydrates, low molecular weight chemical compounds, and analogsand/or mixtures thereof and have binding affinity for one or moreparticles to be detected.

In a further aspect, the invention relates to a method comprisingpositioning multiple particles of a sample within a detection chamber,displacing some of the multiple particles from the detection chamber sothat only a proper subset of the multiple particles remains, opticallydetecting particles of the subset of multiple, particles, and based onthe detected particles, determining a value indicative of the number ofparticles of the subset of particles.

In one embodiment, the method further comprises determining a valueindicative of a number or abundance of particles in the sample based onthe value indicative of the number of particles of the proper subset.Optionally, this determination is further based on a size of a detectionvolume of the detection chamber.

In preferred embodiments, the method further comprises repeating anumber NR times the steps of positioning multiple particles of thesample within the detection chamber and displacing some of the multipleparticles from the detection chamber so that, in each case, only aproper subset of the multiple particles remains, and where NR≧2 and, fora number ND of the NR. repetitions, optically detecting particles of thesubset of multiple particles and, based on the detected particles,determining a value indicative of the number of particles of the propersubset of particles, where ND≧NR.

In further preferred embodiments of the inventive method, repeating thenumber NR times the steps of positioning and displacing comprises, formultiple of the NR repetitions, reintroducing at least some of thedisplaced multiple particles to the detection chamber.

In another preferred embodiment of the invention, displacing some of themultiple particles comprises reducing a volume of the detection chamberwhich, in turn, may comprise reducing a distance between first andsecond walls of the chamber.

In a further embodiment, the inventive method comprises positioningmultiple particles of a sample within a detection chamber, displacingsome of the multiple particles from the detection chamber so that only aproper subset of the multiple particles remains, optically detectingparticles of the subset of multiple particles, and determining thepresence of a target particle among the subset of particles.

In another embodiment, the inventive method comprises positioning afirst multiple of particles of a sample within a detection chamber,reducing a volume of the detection chamber, optically detectingparticles within the detection chamber, based on the detected particles,determining a value indicative of the number of particles present withinthe detection chamber, increasing a volume of the detection chamber,positioning a second multiple of particles of the sample within thedetection chamber, reducing a volume of the detection chamber, and basedon the detected particles, determining a value indicative of the numberof particles present within the detection chamber.

In a further aspect, the invention relates to a device comprising adetection chamber configured to receive a sample comprising multipleparticles, an actuator configured to displace some of the multipleparticles from the detection chamber so that only a proper subset of themultiple particles remains, a detector configured to detect particles ofthe subset of particles, and a processor configured to determine, basedon the detected particles, a value indicative of the number of particlesof the proper subset of particles.

Preferably, the device is configured to operate the actuator toreintroduce at least some of the displaced multiple particles to thedetection chamber and, subsequently, to displace some of the multipleparticles from the detection chamber so that only a second proper subsetof the multiple particles remains and the processor is configured tooperate the detector to detect particles of the second proper subset anddetermine, based on the detected particles, a value indicative of thenumber of particles of the second, proper subset of particles.

The device may further comprise a reservoir capable of receivingparticles displaced from the detection chamber and from which particlescan be reintroduced to the detection chamber.

In one embodiment, the processor may be configured to determine, basedon the detected particles, the presence of a target particle amongparticles of the subset of particles.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic cross-sectional view of an inventive assay devicecomprising an optical detection system.

FIG. 2 is a schematic illustration of an inventive assay employing a.device comprising two displacers located opposite relative to each otheron different sides of the device.

FIG. 3 is a schematic illustration of an inventive assay employing adevice comprising two displacers located at different positions on thesame of the device.

FIGS. 4A and 4B illustrate specific embodiments of an inventive assaydevice or an inventive assay system (A) as well as its mode of operation(B).

FIG. 5 depicts the results of an assay according to the invention fordetermining the number of CD4+ cells in human blood.

FIG. 6 depicts a comparison of CD4+ cell numbers in human blood asobtained by flow cytometry and an assay according to the invention,respectively.

DETAILED DESCRIPTION OF THE INVENTION

In a first aspect, the present invention relates to a device for thequalitative and/or quantitative detection of particles, comprising:

-   -   (a) a reaction chamber formed within a chamber body between a        first surface and a second surface, wherein the second surface        is located opposite to the first surface; and    -   (b) one or more displacers,    -   wherein the distance between the first surface and the second        surface is variable via the one or more displacers at least in        one or more parts of the surface area of the first surface        and/or the second surface.

Within the scope of the present invention, a “reaction chamber” (hereinalso referred to as “reaction space” or “detection chamber” or“chamber”) denotes the space formed within a chamber body between afirst surface and a second surface. The reaction chamber may be of anybasic shape, for example circular, elliptical, quadratic, orrectangular. Preferred reaction chambers of the invention have a cuboidor cylindrical three-dimensional shape. Particularly preferred arereaction chambers being configured as (capillary) channels. Optionally,the reaction chamber may comprise tapering parts. For example, thereaction chamber may taper from the central area to either one or bothterminal regions. The reaction chamber is laterally limited bysidewalls. The second surface is located opposite or substantiallyopposite to the first surface. Preferably, the first and second surfacesare arranged in parallel or substantially parallel to each other.

The distance between the first surface and the second surface is definedas the distance between the side of the first surface of the devicefacing the reaction chamber and the side of the second surface facingthe reaction chamber and is also referred to as thickness of thereaction chamber. According to the present invention, the thickness ofthe reaction chamber is usually at most 1 cm, preferably at most 5 mm,particularly preferably at most 3 mm and most preferably at most I mm.

In some embodiments of the invention the reaction chamber is designed asa capillary gap, which can be tilled by means of capillary forces actingbetween the first and second surfaces. Usually, a capillary gap has athickness of at most 1 mm, preferably of at most 750 μm and particularlypreferably of at most 500 μm. in preferred embodiments of the invention,the capillary gap has a thickness of 300 μm, with a thickness of 200 μmbeing more preferred, and a thickness of 150 μm being particularlypreferred.

In assay devices according to the present invention the distance betweenthe first surface and the second surface is variable. In preferredembodiments, the distance is variable in a range of 0 mm to 1 mm.Further preferred lower limits for the distance between the firstsurface and the second surface are in the range of 50 μm to 200 μm.Further preferred upper limits are in the range of 0.3 mm to 0.5 mm.

The term “displacer” or “actuator”, as used herein, denotes a means thatis suitable to displace a solution within the reaction chamber or in atleast in one or more parts of the reaction chamber of an inventivedevice upon a variation of the distance between the first surface andthe second surface in at least in one or more parts of the surface areaof the first surface and/or the second surface. In other words, adisplacer of the invention may also be defined as a means allowing thevertical movement of the first surface and/or the second surface, or atleast one or more parts thereof, relative to each other. The term “atleast in one or more parts of the surface area”, as used herein, is tobe understood that a variation of the distance between the first surfaceand the second surface via a displacer may not necessarily occur overthe entire surface area of the first surface and/or the second surface(i.e. either one or both of said surfaces as a whole are verticallymoved relative to each other) but may also be locally restricted to atleast one part of the surface area of either one or both of saidsurfaces (i.e. only these one or more parts of the surface area(s) ofthe respective surface(s) is/are moved vertically, relative to eachother, whereas the distance in the remaining one or more parts of thesurface area(s) of the respective surface(s) remains substantiallyconstant). In preferred embodiments of the present invention, thedistance between the first surface and/or the second surface is reduced,particularly preferably by applying pressure to at least a part ofeither one or both of said surfaces.

A displacer according to the invention may constitute an integral partof the first surface or the second surface (herein also referred to as“displacement structure”) or may represent an independent, i.e.self-contained, entity (herein also designated “displacement body”)located outside the reaction chamber. In case a displacer is provided asa displacement structure, i.e. an integral part of the first surface orthe second surface, it may be designed as a convex entity extending tothe inside of the reaction chamber. Such a convex entity may have anyshape that is suitable for causing a displacement of a solution withinthe reaction chamber when performing the inventive method. Examples ofsuch convex shapes include inter alia a dent, a ridge, a buckle, and abulge, with the latter one being preferred. In accordance with itsnature as an integral part of the first surface or the second surfacesuch a displacement structure can be made of the same material as theremainder of the respective surface or at least a part thereof.

In other embodiments of the invention, a displacer constitutes anindependent displacement body not integrated into the first surface orthe second surface but located opposite to either one of said surfaces,wherein the displacer is located at the side of the respective surfacefacing away from the reaction chamber. In some embodiments, thedisplacer is located perpendicular to the respective surface of thereaction chamber. Such a displacement body may have any shape that issuitable, when applied in the invention, to cause a displacement of asolution within the reaction chamber. Examples of such displacementbodies include inter alia a human linger, a rod, a pin, a plunger, aspike, a pole, a tappet, and a stencil, with the latter two beingparticularly preferred.

Within the scope of the invention, the device may comprise one ore moredisplacers, all of which either may be integral parts of the firstsurface and/or the second surface or may be displacement bodies.However, it may also be possible that an inventive device comprises atleast one displacement body and at least one displacement structureintegrated in one of said surfaces. In preferred embodiments of theinvention, the device comprises two displacers. In other embodiments,the device comprises at least three, at least four, at least eight or atleast 12 displacers.

All of the displacers of an inventive device may be integrated in and/orlocated opposite to either of the first surface or the second surface.Alternatively, it is also possible that a device comprises at least twodisplacers that are integrated in and/or located to both of saidsurfaces. In preferred embodiments of the invention, the devicecomprises at least two displacers, which are solely of the displacementbody type located opposite to the first surface and/or the secondsurface. In a particularly preferred embodiment, the device comprisestwo displacers, wherein both displacers are located opposite either tothe first surface or to the second surface. In another particularlypreferred embodiment, the device comprises two displacers as well, butone displacer is located opposite to the first surface, and the otherdisplacer is located opposite to the second surface. In a thirdparticular preferred embodiment, the two displacers located opposite todifferent surfaces are also located opposite relative to each other.

The displacers can be made of any material that is suitable to cause anat least partial displacement of a solution within the reaction chamber,preferably by applying pressure to the first surface and/or the secondsurface giving rise to a variation (i.e. an reduction) of the distancebetween said surfaces. Typically, the displacer(s) is/are made (Wanamorphous material. The term “amorphous material”, as used herein,refers to a solid in which there is no long-range order of the positionsof the atoms, i.e. a non-crystalline material. Examples of suchamorphous materials include inter alia ceramic materials such asaluminum oxide ceramics, glasses such as borofloat glasses, silicone,and synthetic polymers such as polystyrene or polytetrafluorethylene(Teflon™). Optionally, the amorphous material may also be opticallytransparent, i.e. a light-permeable. Examples of suitable transparentmaterials include inter alia glasses or glass-like materials such aswindow glass, borofloat glasses, quartz glasses, topaz glass, orsapphire glass, as well as synthetic polymers such aspolymethytmethacrylate, polycarbonate, polycarbonate, polystyrene, oracryl. In case the inventive device comprises more than one displacer,all of them can be made of the same material or at least one of them canbe made of a different material.

In preferred embodiments of the invention, at least one of the one ormore displacers is made of an elastically deformable material, i.e. amaterial that after having been deformed at least substantially restoresits original shape independently without any further externalmanipulation. Optionally, such elastically deformable materialsaccording to the invention are further preferred to bebiologically/chemically inert (i.e. to have no or a very low reactivitytowards chemical and/or biological reagents), optically transparentand/or not autofluorescent. Examples of such materials include interalia silicone elastomers, polyurethanes, triethyl phosphates, acrylics,and acrylates, with silicone elastomers being particularly preferred.Silicon elastomers are primarily composed of cross-linked siliconepolymers. These polymers have a —Si—O-backbone of the chemical formula[R₂SiO]_(n), where R represents organic side groups such as methyl,ethyl, phenyl or the like, which can be used to crosslink two or morepolymers. By varying the —Si—O— chain lengths, side groups, andcrosslinking, silicones can be synthesized with a wide variety ofproperties and compositions. They can vary in consistency from liquid togel to rubber to hard plastic. The most common type is linearpolydimethylsiloxane. Another group of silicone materials is based onsilicone resins, which are formed by branched and cage-likeoligosiloxanes. In a particularly preferred embodiment, the elasticallydeformable material is selected from the group consisting of a siliconerubber and a silicone oil. Further suitable materials include naturalrubber, that is the rubber extracted of the Para rubber tree (Heveabrasiliensis), as well as further composition rubbers (commonly alsoreferred to as synthetic rubbers) that can be made by polymerization ofa variety of monomers including, e.g., isoprene(2-methyl-1,3-butadiene), 2-chloro-1,3-butadiene, and methyl-propenewith a small percentage of isoprene for cross-linking. Examples ofsuitable composition rubbers include inter alia styrene-butadienerubber, acrylonitrile-butadiene rubber, urethane rubber, polyesterrubber, ehloroprene rubber, butyl rubber, epichlorohydrin rubber, andphosphazene rubber.

In some embodiments of the invention, the first surface and/or thesecond surface comprise(s) a surface area where detection takes place,i.e. a detection area (herein also referred to as “detection zone”).That is the qualitative and/or quantitative detection of the one or morespecies of particles to be analyzed is restricted to a distinct surfacearea of either one or both of said surfaces, for example it may bepossible that detection and/or enumeration of the particles will takeplace only in the central section of the first surface and/or the secondsurface, probably due to spatial constraints of the detection systemused. Preferably, the volume of the section of the reaction chamber thatis limited by the detection area(s) of the first surface and/or thesecond surface is known to allow for quantitative analyses. In someembodiments of the invention, the “detection area” between the firstsurface and the second surface is designed as a capillary gap that ispreferably located in the central part of the reaction chamber, whereinthe distance between said surfaces in this area may optionally besmaller than the distance in the remaining parts of the reactionchamber.

In other embodiments of the invention, at least one or more parts of thefirst surface and/or the second surface is/are elastically deformable,That is, at least one or more parts of the respective surface(s) is/aremade of an elastically deformable material, for example an elasticmembrane, as described above in connection with the one or moredisplacers. A particularly preferred elastic membrane according to theinvention is made of silicone rubber. Thus, in one embodiment the entiresurface area of the first surface and/or the second surface can be madeof an elastically deformable material. In one embodiment of theinvention, the whole reaction chamber is elastically deformable, thatis, the first surface, the second surface, and the lateral side walls.Preferably, the at least one or more elastically deformable parts of thefirst surface and/or the second surface are located opposite to the oneor more displacers. Accordingly, all surface areas of an inventivedevice located opposite of a displacer may be elastically deformable.However, it is also possible that at least one such surface area locatedopposite to a displacer is not elastically deformable. In one preferredembodiment of the invention, the at least one or more elasticallydeformable parts of the first surface and/or the second surface aredifferent from the respective surface area(s) where detection take(s)place.

According to the present invention, the first surface and the secondsurface can be made of the same material or of different materials.Furthermore, it is also possible that the first surface and/or thesecond surface comprise(s) surface areas made of different materials asthe remainder of the respective surface area. For example, one surfacearea of the first surface and/or the second surface, such as a central,optionally rectangular, surface area (i.e. a “window”), is made of atransparent material, whereas the remainder of the respective surfacearea (i.e. the “border”) is made of a non-transparent material.Preferably, such a “window” of transparent material is located withinthe surface region where detection takes place.

At least a part of the first surface and/or the second surface of deviceaccording to the invention may be made of an amorphous material. Theterm “amorphous material”, as used herein, refers to a solid in whichthere is no long-range order of the positions of the atoms, i.e. anon-crystalline material. Examples of such amorphous materials includeinter alia ceramic materials such as aluminum oxide ceramics, glassessuch as borofloat glasses, silicone, and synthetic polymers such aspolystyrene or polytetrafluorethylene (Teflon™).

In preferred embodiments of the invention, at least a part of the firstsurface and/or the second surface is/are made of a transparent material,i.e. a light-permeable material. Examples of suitable transparentmaterials include inter alia glasses or glass-like materials such aswindow glass, borofloat glasses, quartz glasses, topaz glass, orsapphire glass, as well as synthetic polymers such aspolymethyl-methaerylate, polycarbonate, polycarbonate, polystyrene, oracryl.

A device according to the present invention may further comprise amicroarray (herein also referred to as “array” or “array element”) beingdisposed on the first surface and/or the second surface of the reactionchamber. As used herein, a “microarray” denotes a defined spatialarrangement (layout) of capture molecules (e.g., one or more species ofprobe molecules or a substance library) on a support (also referred toas substrate), wherein the position of each molecule within themicroarray is determined separately. Preferably, the microarraycomprises defined sites or predetermined regions, i.e. so-called arrayelements or spots, which may be arranged in a particular pattern,wherein each array element preferably comprises only one species ofcapture molecules. The arrangement of the capture molecules on thesupport can be generated by means of covalent or non-covalentinteractions. Suitable substrates for microarrays include inter aliamicroscope slides, wafers or ceramic materials. However, the capturemolecules may also be directly immobilized on the first surface and/orthe second surface.

The device according to the present invention may further comprise oneor more means, which, when the distance between the first surface andthe second surface is reduced, allow keeping the volume of the reactionchamber essentially constant. That is, compensation zones (or“reservoirs”) are provided to which any liquid material being present inthe reaction chamber between the first surface and the second surfacecan be displaced when the distance between said surfaces is reduced. Theterm “essentially constant”, as used herein, is to be understood thatupon a reduction of the distance between the first surface and thesecond surface the volume of the reaction chamber in the “compressed”state has not to be exactly the same as its original volume in the“uncompressed” state. Preferably, the volume of the reaction chamber inthe “compressed” state is at least 90% of the volume in the“uncompressed” state, particularly preferable at least 95%. In someembodiments of the invention, any reagents required for performing theinventive methods such as agents comprising detectable moieties (i.e.labels) or buffers are provided in the compensation zones or reservoirs,preferably in dry form (e.g. in lyophilized form as powdem, granules orpellets).

Preferably, this is accomplished by providing a reaction chamberlaterally delimited by sidewalls made of an elastic material. Accordingto the present invention, one or more lateral sidewalls can be made ofan elastic material. A particularly preferred elastic material issilicone rubber.

In another embodiment, the inventive device further comprises a chamberbody. The term “chamber body”, as used herein, is understood to denotethe solid body surrounding the reaction chamber, which is formed by thefirst surface, the second surface, and the lateral sidewalls.

The first surface, the second surface, and/or one or more of the lateralsidewalls may be integral part(s) of the chamber body. That is, therespective surface(s) being an integral part of the chamber body is/aremade of the same material as the chamber body. Alternatively, one ormore of the first surface, the second surface, and/or one or morelateral sidewalls, respectively, may be made of another material thanthe chamber body. Within the scope of the present invention, it is thuspossible that all four surfaces defining the reaction chamber are madeof the same material, that two or three surfaces are made of the samematerial, whereas the remaining surface(s) is (are) made of differentmaterial(s), or that each surface is made of different materials.

The first surface and/or the second surfaces and/or one or more lateralsidewalls delimiting the reaction chamber may further comprise one ormore openings, which may be lockable and/or scalable, and which may beused for the direct introduction of a sample to be analyzed as well asany additional reagents, detection agents or the like that mayoptionally also be required for performing the method of the invention.Alternatively, such openings may also be used for the attachment of anyadditional (supplementary) modules of the device that have not beendesigned as integral parts of the chamber body, such as inter aliafilling units, processing units, temperature control units, specific(i.e. complex) detection units, and waste containers. A connectionbetween the reaction chamber and one or more of such additional modulesmay be achieved inter alia by using one or more rigid or flexible tubes,nozzles, cannulae, needles or the like, which may be attached to thereaction chamber and the additional module, respectively, inter alia bymeans of press-fit (also referred to as “Luer system”) or twist-onfitting (also referred to as “Luer-lock system”) with the latter onebeing preferred. Both systems are well established in the art andcommercially available.

The chamber body is preferably made at least in part of an amorphousmaterial, in particular of a transparent material. Suitable materialsinclude inter alia glass, synthetic materials such as Macrolon™, nylon,potymethylmethaerylate, Teflon™, and metals such as high-grade steel,aluminum, and brass. In some embodiments, the chamber body is made orelectrically conductive material, which is preferably selected from thegroup consisting of polyamide with 5 to 30% carbon fibers, polycarbonatewith 5 to 30% carbon fibers, polyamide with 2 to 20% stainless steelfibers, and polyphenylensulfide with 5 to 40% carbon fibers.

The devices according to the present invention are typically operated asa single (i.e. individual) entity. However, it may also be possible to,assemble two or more such devices to a multipart system comprisingseparate reaction chambers in order to perform multiple assays of onesample in parallel wherein a device as described above corresponds to apart or unit or entity of the multipart system. Thus, in a furtheraspect, the present invention relates to a system, comprising at leasttwo parts, wherein each of the at least two parts comprises a reactionchamber formed within a chamber body between a first surface and asecond surface, wherein the second surface is located opposite to thefirst surface; and one or more displacers, wherein the distance betweenthe first surface and the second surface is variable via the one or moredisplacers at least in one or more parts of the surface area of thefirst surface and/or the second surface. The reaction chambers of the atleast two parts of the system may be in communication with each other.

The term “in communication with each other”, as used herein, denotes anyinterconnection between the individual reaction chambers, eitherdirectly or indirectly via an additional means such as a common sampleintroduction passage, filling unit, processing unit or the like.However, as used herein, the terra does not necessarily mean that, afterintroducing a sample, the reaction chambers are in permanent fluidcommunication with each other.

In a preferred embodiment, the system further comprises a sampleintroduction passage which is in communication with each of the reactionchambers of the at least two devices. The sample introduction passage(herein also referred to as “sample loading zone” or “sample area”) maycomprise one or more lockable and/or sealable openings, as describedabove, for introducing the sample to be analyzed. The sampleintroduction passage may be configured as a chamber from which therespective reaction chambers branch off. The openings connecting thesample introduction passage and the reaction chambers may be lockableand/or sealable as well. In particular, it is preferable to prevent,after introducing a sample into the separate reaction chambers of such amultipart system, a backflow of liquid from a reaction chamber into thecommon sample introduction passage which would result in a mixing ofsample solutions from different reaction chambers comprising variablereagents (such as capture molecules, labels and the like) and thus aninterference with proper detection of the multiple individual assays tobe performed in parallel.

Thus, in a further preferred embodiment of the invention, the systemfurther comprises one or more means that allow for a transient fluidcommunication between the at least two reaction chambers. It isparticular preferred to allow only for a unidirectional fluidcommunication from the sample introduction passage to the separatereaction chambers but not backwards. This may preferably be achieved bythe provision of one-way valves at the connections between the sampleintroduction passage and the reaction chambers which prevent backflow ofthe sample from the reaction chambers.

In preferred embodiments of the invention, the device further comprisesa detection system connected to the reaction chamber. Preferably, thedetection system is positioned opposite to the first surface and/or thesecond surface, optionally in a particular surface region wheredetection takes place.

The selection of a suitable detection system depends on severalparameters such as the optional presence of additional agents (e.g. dyesor labels) used for detection or the kind of particles to be detected.Various optical and non-optical detection systems are well establishedin the art. A general description of detection systems that can be usedwith the invention can be found, e.g., in Lottspeich, F., and Zorbas H.(1998) Bioanalytik, Spektrum Akademischer Verlag, Heidelberg/Berlin,Germany, in particular in chapters 23.3 and 23.4.

In a preferred embodiment of the invention, the detection system is anoptical detection system. In general, performing the method according tothe present invention does not require the use of sophisticatedinstrumentation but rather involves simple detection systems, preferablybased on the measurement of parameters such as fluorescence, opticalabsorption, resonance transfer, and the like.

Particularly preferred detection systems according to the invention arebased on apsorption measurements such as turbidimetry and nephelometry(see for review, e.g., Tiffany, O. (1986) Fluorometty, nephelometry, andturbidimetty, in: Tietz, N. O. (ed.). Textbook of Clinical Chemistry. WBSaunders Co., Philadelphia, Pa., pp. 78-97), which may be achieved withphotometric devices established in the art, Both methods allow thedetermination of the “cloudiness” or turbidity in a solution based uponmeasurement of the effect of this turbidity upon the transmission orscattering of light, respectively. Turbidity in a liquid is caused bythe presence of particles suspended therein. If a beam of light ispassed through a turbid sample, its intensity is reduced. Turbidimetryrefers to the measurement of unscattered light, i.e. light that istransmitted through a turbid solution of particles that can be performedusing a standard photometer. Nephelometry, on the other hand, is themeasurement of (side) scattered light. This technique requires a specialinstrument, where the detector is set at an angle to the incident lightbeam.

Also preferred for use in the present invention are detection systemsbased on the comparison of the fluorescence intensities of spectrallyexcited particles labeled with fluorophores. Fluorescence is thecapacity of particular molecules to emit their own tight when excited bylight of a particular wavelength resulting in a characteristicabsorption and emission behavior. In particular, quantitative detectionof fluorescence signals is performed by means of modified methods offluorescence microscopy (for review see, e.g., Lichtman, J. W., andConchello, J. A. (2005) Nature Methods 2, 910-919; Zimmermann, T. (2005)Adv. Biochem. Eng. Biotechnol. 95, 245-265). Thereby, the signalsresulting from light absorption and light emission, respectively, areseparated by one or more filters and/or dichroites and imaged onsuitable detectors. Data analysis is performed by means of digital imageprocessing.

Image processing may be achieved with several software packages wellknown in the art (such as Mathematica Digital Image Processing, EIKONA,or Image-PRO). Since the present invention is intended to be performedwithout any removal and/or replacement of solutions from the reactionchamber (that is, detection is performed in the presence of any unboundlabels giving rise to an unspecific signal background), it is preferredto use an image processing software allowing for an efficient andaccurate correction of background noise. Particularly preferably,detection of specific signals is not only achieved by measuringdifferences in signal intensities but also by concomitantly taking intoaccount additional parameters such as the “shape” of the signal to bedetected, which is again determined by the shape of the particles to bedetected. For example, a circular particle such as a cell of circularshape that is labeled with a fluorescent antibody will result in a“circular signal” in the image recorded during optical detection. Apreferred software suitable for such purposes is the lconoclust software(Clondiag Chip Technologies GmbH, Jena, Germany).

Another optical detection system that may be used in the presentinvention is confocal fluorescence microscopy, wherein the object isilluminated in the focal plane of the lens via a point light source.Importantly, the point light source, object and point light detector arelocated on optically conjugated planes. Examples of such confocalsystems are described in detail, e.g., in Diaspro, A. (2002) Confocaland 2-photon-microscopy: Foundations, Applications and Advances,Wiley-Liss, Hobroken, N.J. The fluorescence-optical system of thepresent invention is particularly preferred to represent a fluorescencemicroscope without an autofocus, for example a fluorescence microscopehaving a fixed focus.

In alternative devices according to the present invention means forperforming an electrochemical detection of the analytes are provided,for example by measuring the alteration of redox potentials viaelectrodes connected to the first surface and/or the second surface(see; e.g., Zhu, X. et al. (2004) Lab Chip. 4; 581-587) or by cyclicvoltometry (see, e.g., Liu, J. et al. (2005) Anal. Chem. 77, 2756-2761;and Wang, J. (2003) Anal. Chem. 75, 3941-3945). Furthermore, it is alsopossible to provide means for performing an electric detection, forexample by impedance measurement (see, e.g., Radke, S. M. et al. (2005)Biosens. Bioelectron. 20, 1662-1667).

Typically, the devices and systems according to the present inventionare self-contained. That is, they do not necessarily require removaland/or replacement of the sample and/or any other reagents in thereaction chamber while performing an assay. Thus, preferred embodimentsof devices and systems of the invention only comprise a sample inletport but no outlet port.

In a further aspect, the present invention provides a method for thequalitative and/or quantitative detection of particles, comprising:

-   -   (a) positioning a sample supposed to comprise, one or more        species of particles to be detected in a reaction chamber,    -   (b) displacing at least a part of the sample within the reaction        chamber via the one or more displacers; and    -   (c) detecting/determining a value indicative for the presence        and/or number of one or more species of particles.

In preferred embodiments of the invention, “positioning a sample in areaction chamber” comprises introducing said sample into the reactionchamber of an inventive device or system as described above.

In a further particular preferred embodiment of the invention, themethod comprises:

-   -   (a) positioning a sample comprising multiple particles in a        reaction chamber;    -   (b) displacing a subset of said multiple particles within the        reaction chamber via the one or more displacers; and    -   (c) determining one or more values indicative for the number of        the subset of particles displaced in step (b).

Optionally, the method further comprises:

-   -   (d) calculating the total number of the multiple particles in        the reaction chamber from the one or more values obtained in        step (c).

In some embodiments of the invention, the quantitativedetection/determination in step (c) comprises the counting of one ormore species of particles in a sample, for example the enumeration ofcells in a biological sample.

The term “sample”, as used herein, refers to a liquid which is to beanalyzed by using a device according to the present invention, and whichis supposed to comprise one or more species of particles to be detected.Preferably, the sample to be analyzed is a biological sample. Examplesof liquid samples that can be analyzed using the invention include interalia organic and inorganic chemical solutions, drinking water, sewage,human and non-human body fluids such as whole blood, plasma, serum,urine, sputum, salvia or cerebrospinal fluid, cellular extracts fromanimals, plants or tissue cultures, prokaryotic and eukaryotic cellsuspensions, phage preparations and the like. The sample may furthercomprise one or more additional agents such as diluents, solvents orbuffers that may result from an optional purification and/or processingof the sample prior to its introduction (positioning) into the reactionchamber.

The sample to be analyzed may comprise one or more species of particlesto be detected when performing the present invention. The term“particle”, as used herein, denotes any entity having a specific bindingbehavior and/or a characteristic reactivity, which enables its detectionby performing the method of the invention. Accordingly, the term“particle” is not only to be construed in a literal sense as referringto “true” particles such as cells or viruses but also to be understoodas comprising molecules to be detected, in particular (biological)macromolecules such as nucleic acids, proteins, lipids, andcarbohydrates as well as analogs and/or mixtures thereof having bindingaffinity to any (synthetic) particles present in or added to a sample tobe analyzed.

The term “species”, as used herein in connection with the termparticles, refers to a particular type of particle, i.e. a specific typeof cells or a specific phage, for example. Accordingly, the term “one ormore species” denotes one or more different types of particles such asone or more different cell types.

“True” particles according to the present invention include naturallyoccurring as well as synthetic particles. Examples of such naturallyoccurring particles include inter alia prokaryotic cells (e.g. bacterialcells such as Escherichia coli or Bacillus subtilis), eukaryotic cells(e.g. yeast cells such as Saccharomyces cerevisiae, insect cells such asSf9 or High 5 cells, immortalized cell lines such as HeLa or Cos cells,and primary cells such as mammalian blood cells) or viruses (e.g. phageparticles such as M13 or T7 phage). Examples of synthetic particleinclude inter alia (paramagnetic) polystyrene beads and latex beads,optionally be coated with one or more species of molecules to bedetected by the present invention, in particular one or more species of(biological) macromolecules such as nucleic acids, proteins, lipids orcarbohydrates.

The sample to be analyzed may be introduced directly into the reactionchamber via one or more openings, which may be lockable and/or sealable,being present in the first surface, the second surface and/or one ormore lateral sidewalls. The sample may be transferred, optionally alongwith additional reagents, into the reaction chamber by using a suitablepressure-generating means, for example, a pipette, a syringe or anautomated unit, which may be, for example, a functional unit of aprocessing apparatus. Alternatively, the sample may also be introducedinto the reaction chamber by capillary force without any externalmanipulation, for example by placing the sample immediately adjacent toone of the openings being present in any of the surfaces defining thereaction chamber.

In a specific embodiment of the inventive method, the sample isintroduced into the reaction chamber by means of negative pressurecaused by operating the one or more displacers. In this embodiment,initially, i.e. before adding the sample, the distance between the firstsurface and the second surface in at least in one or more parts of thereaction chamber, or in other words, in at least in one or more parts ofthe surface area of the first surface and/or the second surface isreduced by applying pressure towards the first and/or second surface viathe one or more displacers. Preferably, the distance is reduced to avalue of zero or almost zero. Then, said reduced distance isre-increased, preferably to the initial value, by resetting thedisplacer (i.e. by moving it in backwards direction) thus causing anegative pressure within the reaction chamber which, in turn, allows forintroducing the sample into the reaction chamber.

The method of the present invention is intended to be performed withoutthe requirement to remove and/or replace the sample and/or any otherreagents in the reaction chamber while performing the method. Inparticular, it is an advantage of the inventive method that no washingor rinsing steps that would require such removal/replacement arenecessary, for example in order to improve the signal-to-noise ratio ofthe detection method used. However, some applications may require theintroduction of additional reagents into the reaction chamber such asone or more agents comprising any labels in order to allow furtherdetection of the particles of interest. Such additional solutions mayalso be directly introduced into the reaction chamber, as describedabove, either before introducing the sample or concomitantly with thesample or after the sample has been introduced into the reactionchamber. In preferred embodiments, the additional reagents are providedwithin the device (e.g., in the compensation zones) before adding thesample, particularly in lyophilized/dry form such as powders, granulesor pellets.

Alternatively, introducing the sample to be analyzed, and optionally offurther reagents, may also be possible in an indirect manner by means ofone or more filling units.

Within the scope of the present invention, a “filling unit” denotes ameans for filling the reaction chamber which may be an integrated partof the device of the invention or it may be designed as a separate partthat can be attached to the reaction chamber for filling the same anddetached after use. Any container that is capable of holding a liquidsample to be analyzed in the invention and that can be (reversibly)connected to the reaction chamber may be used as filling unit. Aconnection between reaction chamber and filling unit may be achievedinter alia by means such as tubes, nozzles, cannulae, needles or thelike, as already described above. A given sample can be introduced intoone or more lockable and/or scalable openings of the filling unit in thesame way as described above for the direct introduction into thereaction chamber.

In special embodiments, one or more cannulae are used for connecting afilling unit to the reaction chamber of the device. The cannulae usedpenetrate the lock and/or seal of one or more of the openings comprisedin the reaction chamber. Preferred cannulae used in the invention aremade of high-grade steel or of synthetic polymers and usually have adiameter of 0.05 mm to 2 mm. Preferably, two cannulae are arranged insuch a way that one is used for introducing the sample into the reactionchamber and the other one for taking up excess gaseous material and/orsurplus liquids from the reaction chamber (for a detailed descriptionsee also the International Patent Application WO 01/02094, whoserelevant contents are herewith explicitly referred to).

The filling unit may comprise an integrated or a detachable separatewaste container, which serves for taking up surplus media from thereaction chamber. Optionally, the waste container comprises with afurther gaseous, liquid, or solid filler medium such as inter ciliacellulose, filter materials, and silica gels, which binds the surplussubstances reversibly or irreversibly. Furthermore, the waste containermay comprise one or more air vents or may be provided with a vacuum inits interior for improving the transfer of surplus material to the wastecontainer.

The filling unit may further comprise mechanical means ensuring that itaccurately fits the respective attachment site of the reaction chamber,i.e. that the filling unit is exactly positioned relative to thereaction chamber to allow connecting the filling unit to the reactionchamber via one or more cannulae, nozzles or the like at preferred sitessuch as the lockable and/or scalable openings. Examples of suchmechanical means include inter alia specifically designed snap fits orspring locks. Preferably, the mechanical means allow detaching thefilling unit after introducing the sample and any optional reagents intothe reaction chamber.

One advantage of the present invention refers to the fact that samplevolumes of less than 10 μl can be analyzed. Typically, sample volumesare in a range of 1 to 1.000 μl, preferably in a range of 1 to 100 μl,more preferably in a range of 1 to 25 μl, and most preferably in a rangeof 1 to 5 μl.

The samples to be analyzed can be introduced into the reaction chamberwithout any further purification, since the inventive device and methodare specifically designed to allow the qualitative and/or quantitativedetection of any particles in a given sample without the requirement toperform washing and rinsing steps. However, in some cases it might bepreferable to purify the sample, at least partially, for example inorder to remove any crude contaminations that would otherwise interferewith further detection. Such (partial) purification of the sample can beaccomplished in different ways, for example by filtration of the samplebefore introducing it into the reaction chamber.

Furthermore, it may be required to dilute a sample to be analyzed due toa comparably high viscosity that would otherwise interfere with thediffusion of the sample throughout the reaction chamber of the inventivedevice. Dilution of the sample can be easily achieved by adding adiluent to the sample. Examples of suitable diluents include inter aliawater, organic and inorganic solvents, phosphate-buffered saline and thelike. The diluent may be added before introducing of the sample into thedevice or may be directly added into the tilling unit and/or thereaction chamber, as described above.

After the sample, and optionally any additional reagents, have beenintroduced into the reaction chamber or have been transferred from theone or more filling units into the reaction chamber, the sample mayoptionally be incubated in the reaction chamber for a given period oftime to allow proper diffusion throughout the reaction space. Typically,the incubation period is in the range of I s to 30 min, preferably inthe range of 10 s to 15 min, and particularly preferably in the range of30 s to 10 min.

In some preferred embodiments, the method according to the presentinvention further comprises introducing one or more agents eachcomprising one or more detectable moieties into the reaction chamber ofthe device before performing step (b). That is, the agents comprisingone or more detectable moieties may be introduced into the reactionchamber before introducing the sample, concomitantly with the sample, orafter the sample has been introduced either directly or via a tillingunit, as described above.

The term “agent comprising one or more detectable moieties”, as usedherein, refers to any compound that comprises one or more appropriatechemical substances or enzymes (i.e. one or more “moieties”), whichdirectly or indirectly generate a detectable compound or signal in achemical, physical or enzymatic reaction. Such an agent may thus benecessary for or will facilitate detection of one or more species ofparticles of interest by being capable of forming interactions with saidparticles.

As used herein, the term is to be understood to include both detectablemarkers as such (also referred to as “labels”) as well as any compoundscoupled to one or more such detectable markers.

In preferred embodiments, the one or more agents are selected from thegroup consisting of nucleic acids, peptides, protein domains, proteins,carbohydrates, low molecular weight chemical compounds, and analogsand/or mixtures thereof.

Examples of nucleic acids that can be used as in the present inventioninclude naturally occurring nucleic acids such as deoxyribonucleic acid(DNA) or ribonucleic acid (RNA) as well as nucleic acid analogs such asinter alia peptide nucleic acids (PNA) or locked nucleic acids (LNA).Specific examples of naturally occurring nucleic acids include DNAsequences such as genomic DNA or cDNA molecules as well as RNA sequencessuch as hnRNA or mRNA molecules or the reverse complement nucleic acidsequences thereof. Such nucleic acids can be of any length and can beeither single-stranded or double-stranded molecules, withsingle-stranded molecules being preferred. Typically, such nucleic acidsused in the invention are 10 to 1000 bases in length, preferably of 15to 500 bases, more preferably 20 to 100 bases and particularlypreferably of 20 to 40 bases.

Peptides, protein domains or proteins that can be used as agentsaccording to the present invention comprise naturally occurring as wellas artificially designed molecules, for example by means of recombinantDNA technology or via chemical synthesis. Methods for the design andpreparation of such proteinaceous molecules are well established in theart (see, for example, Sambrook, J. et al. (1989) Molecular, Cloning: ALaboratory Manual, 2nd ed., Cold Spring Harbor. Laboratory Press, ColdSpring Harbor, N.Y.). Typically, peptide agents of the invention are 2to 200 amino acids in length, preferably 2 to 100 amino acids, morepreferably of 5 to 50 amino acids, and particularly preferably of 10 to25 amino acids.

The term “protein domain”, as used herein, refers to a part of apolypeptide sequence that is defined with regard to the specificfunction it exhibits, such as ligand binding or catalytic activity.Preferred examples of such protein domains are inter alia Fab-fragmentsof antibodies, the ligand-binding domains of cellular receptors such asG-protein coupled receptors, receptor tyrosine kinases or nuclearreceptors, and the carbohydrate-binding domain of lectins.

Examples of carbohydrates that can be used as agents in the presentinvention include monosaccharides such as glucose or fructose,disaccharides such as lactose or sucrose, as well as oligosaccharidesand polysaccharides such as starch, with monosaccharides beingpreferred.

The term “low molecular weight chemical compound”, as used herein,denotes an molecule, preferably an organic molecule, comprising at leasttwo carbon atoms, but preferably not more than seven rotatable carbonbonds, having a molecular weight in the range between 100 and 2.000Dalton, preferably between 100 and 1.000 Dalton, and optionallyincluding one or two metal atoms. Examples of such molecules includeinter alia imidazoles, indoles, isoxazoles, oxazoles; pyridines,pyrimidines, and thiazoles.

Detectable markers or labels that may be used according to the inventioninclude any compound, which directly or indirectly generates adetectable compound or signal in a chemical, physical or enzymaticreaction. Preferably, the labels can be selected inter alia from enzymelabels, colored labels, fluorescent labels, chromogenic labels,luminescent labels, radioactive labels, haptens, biotin, metalcomplexes, metals, and colloidal gold, with fluorescent labels beingparticularly preferred. All these types of labels are welt establishedin the art. A preferred example of a physical reaction that is mediatedby such labels is the emission of fluorescence or phosphorescence uponirradiation or excitation or the emission of X-rays when using aradioactive label. Alkaline phosphatase, horseradish peroxidase,β-galactosidase, and β-lactamase are examples of enzyme labels, whichcatalyze the formation of chromogenic reaction products, and which maybe used in the invention. A preferred enzyme label is horseradishperoxidase, especially along with a substrate selected from the groupconsisting of 3-amino-9-ethylcarbazole, 4-chloro-1-naphthol,3,3′-diaminobenzidine, and 3,3′,5,5′-tetramethyl-benzidine, with thelatter one being particularly preferred.

In preferred embodiments of the inventions, the one ore more agentscomprising one or more detectable moieties are directly coupled to oneor more species of particles to be detected, wherein it is to be notedthat in the invention such coupling will typically not occur to theparticles “as a whole” but to one or more species of particularmolecules, preferably macromolecules such as a nucleic acid or aprotein, on the surface of said particles. Such a “surface molecule” maybe a molecule naturally occurring on the surface of cell (e.g. a cellsurface receptor such as a G-protein coupled receptor or a particularcarbohydrate moiety) or may be artificially coated to the surface of aparticle, for example a nucleic acid molecule (also referred to as“capture molecule” or “molecular probe”) coupled to a paramagnetic bead,for example via an biotin/avidin-linkage. The labeling reaction, i.e.the attachment of one or more detectable moieties to an agent of theinvention, may be performed outside the inventive device, i.e. beforeintroducing the sample, or directly in the device, optionally in thefilling unit already described above. Labeling can be achieved bymethods well known in the art (see, for example, Sambrook, J. et al.,supra; and Lottspeich, F., and Zorbas H., supra).

Preferably, the one or more agents each comprising one or moredetectable moieties have binding affinity for the particular “surfacemolecule” (or to the particle as such), to which they bind. Examples ofsuch agents include inter alia antibodies as well as fragments thereof(e.g. Fab fragments), antibody-like molecules (e.g. anticalins), andDNA- or RNA-binding proteins as well as fragments thereof. Suitableantibodies or antibody fragments to be used in the invention includeboth primary antibodies which are raised against the particular analyteto be detected and secondary antibodies which are raised againstimmunglobulin G of the animal species in which the primary antibody hasbeen raised. Labeling is accomplished by coupling the agents to one ormore detectable markers as described above.

In another preferred embodiment, one or more agents each comprising oneor more detectable moieties and the “surface molecules” on the particlesto be detected (or the particle as such) are allowed to form molecularinteractions with each other, while being present in the reactionchamber. Finally, these molecular interactions are detected using anappropriate detection method.

After having introduced the sample, optionally along with any additionalreagents, into the reaction chamber at least a part of the sample isdisplaced within the reaction chamber via the one or more displacers.The term “displaced”, as used herein, is to be understood that thesample is moved within the reaction chamber by varying, preferably byreducing, the distance between the first surface and the second surfaceat least in one or more parts of the surface area of the first surfaceand/or the second surface via the one or more displacers, preferably byapplying pressure to either one or both of said surfaces or at least oneor more parts thereof. Accordingly, it is within the scope of thepresent invention either to vary the distance between the first surfaceand the second surface throughout the entire surface area of saidsurfaces or to vary the distance in only one part of the surface areasuch as at one terminal end of the reaction chamber or to vary thedistance between the first surface and the second surface in at leasttwo distinct parts of the surface area such as at both terminal ends ofthe reaction chamber.

A variation, preferably a reduction, or the distance between the firstsurface and the second surface is accomplished by vertically moving thefirst surface and/or the second surface relative to each other via theone or more displacers. The term “vertical movement”, as used herein,denotes a movement or either one or both surfaces of the deviceperpendicular or substantially perpendicular to their respective surfaceareas, thus resulting in a variation of the distance between them. Avariation of the distance between the first surface and the secondsurface can be achieved by vertically moving either one of the twosurfaces in either direction or by moving both surfaces simultaneouslyin opposite directions. Accordingly, a reduction of the distance betweenthe first surface and the second surface of the device can be achievedeither by moving the first surface or at least one or more parts thereoftowards the second surface, by moving the second surface or at least oneor more parts thereof towards the first surface or by moving bothsurfaces or at least one or more respective parts thereof towards eachother. Vice versa, an increase of the distance between the first surfaceand the second surface of the device can be achieved either by movingthe first surface away from the second surface, by moving the secondsurface away from the first surface or by moving both surfaces away fromeach other. In particular, it is preferred to vary the distance betweenthe first surface and the second surface by applying pressure and/ortraction to either one or to both surfaces via said one or moredisplacers. Within the scope of the present invention, it is possible toapply pressure and/or traction concomitantly via all displacers presentin a given device or via only at least one of them, whereas the other atleast one displacers remain unused. For example, in a device of theinvention comprising two displacers it is thus possible to applypressure and/or traction to at least one part of the first surfaceand/or second surface concomitantly via both displacers or with only oneof them in order to displace the sample within the reaction chamber.

Thus, in view of the different types of displacers defined abovedifferent modes concerning the variation, preferably reduction, of thedistance between the first surface and the second surface (in thefollowing the terms “first surface” and/or “second surface” are to beunderstood also to refer to at least in a part of the respective surfacearea) are conceivable, finally resulting in the displacement of at leasta part of the sample within the reaction chamber.

First, in case a displacer constitutes an integral part of the firstsurface or the second surface (e.g. a displacement structure designed asa convex entity extending to the inside of the reaction chamber) forreducing the distance between said surfaces it is possible to move therespective surface comprising the displacement structure verticallytowards the opposite surface, to move the opposite surface verticallytowards the surface comprising the displacement structure, or to moveboth surfaces vertically relative towards each other. Optionally, thesurface opposite of the “integrated” displacement structure alsocomprises, a displacer at the respective (i.e. opposite) location, whichmay be integrated into the surface as well or may represent adisplacement body, which will be operated as described below. Verticalmovement of the respective surface(s) may either be achieved by one ormore displacers of the displacement body type or by one or moreadditional means allowing the vertical movement of said surfaces. Suchmeans may be integrated in or attached to the first surface and/or thesecond surface of the device or may comprise an independent entity. insome embodiments, the one or more means are selected from the groupconsisting of a human finger, a rod, a pin, a tappet, and a screw. Ifthe device is integrated into an automated processing system, one ormore means such as a stamp or a plunger connected to the reactionchamber may be used to apply pressure on the first surface and/or thesecond surface. Another means for applying pressure to the surfaces isto simply press them together in one's hand.

Second, in case a displacer is present in form of a displacement bodyfor reducing the distance between said surfaces pressure may be appliedto the respective surface, opposite to which the displacer is located,by moving said displacer towards the surface, which may optionally bemade of elastically deformable material at least in this part of thesurface area. The (elastically) deformation of the respective surface,i.e. a “movement” of the surface in vertical direction, via thedisplacer then results in a reduction of the distance between the firstsurface and the second surface. Optionally, a second displacer may belocated opposite to the above-mentioned displacement body on the other“side” of the reaction chamber. This second displacer may either be adisplacement structure integrated into the respective surface or may beanother displacement body located opposite to it. Vertical movement ofthe second displacer occurs in reverse direction than that of the firstdisplacer.

Reducing the distance between the first surface and the second surfaceof the reaction chamber at least in one or more parts of the surfacearea(s) results in a concomitant (spatially restricted) reduction of thereaction space at the respective sites at which the distance has beendecreased. Accordingly, the sample to be analyzed becomes at least inpart successively displaced from these sites and is “moved” within thereaction chamber, preferably to the compensation zones that allowkeeping the volume of the reaction chamber essentially constant.

After having reduced the distance between at least one or more parts ofthe first surface and the second surface it is possible to immediatelyperform the detection, as will be described below.

The term “detecting/determining a value indicative for the presenceand/or number of one or more species of particles”, as used herein,refers to the determination of parameters such as electricalconductivity, redox potential, fluorescence intensity or bioluminescencethat allow for qualitative and/or quantitative measurements of givenparticles in a sample. Within the scope of the present invention, only asingle of these parameters may be determined but it is also possible todetermine more than one parameter (e.g., electrical conductivity and theintensity of a fluorescence signal caused by a suitable label), eitherconcomitantly or consecutively.

Preferably, the detection is performed in the part of the reactionchamber that is located between the detection area(s) of the firstsurface and/or the second surface, as described above. This part of thereaction chamber is also referred to as “detection zone”. Forquantitative measurements, i.e. the counting of particles, it is thuspreferred to employ a device comprising a reaction chamber and adetection zone having known volumes, respectively.

However, at this stage of the method it is preferred to re-increase thedistance between the at least one or more parts of the first surface andthe second surface of the reaction chamber. It is particularly preferredto restore the original distance, i.e. the distance before thereduction, between the first surface and the second surface of thereaction chamber. This may be achieved by vertical movement of the atleast one displacement structures and optionally also of the meansallowing vertical movement of said surfaces, as described above, but inreverse direction, i.e. by discontinuing the application of pressure tothe respective surfaces or surface areas. Optionally, the re-increase ofthe distance between the First surface and the second surface that hasbeen reduced in a given surface area by applying pressure via a firstdisplacer may be improved by reducing the distance in another surfacearea by applying pressure via a second displacer. Preferably, bothdisplacers represent displacement bodies, which may be located oppositeto the same surface, i.e. either the first surface or the secondsurface, or opposite to different surfaces. Re-increasing the distancebetween the two surfaces in the at least one or more surface areas ofthe first surface and/or the second surface results in a concomitant(optionally spatially restricted) increase in reaction space between thetwo surfaces. Furthermore, the sample having been displaced within thereaction space, preferably in the compensation zones provided, will nowdiffuse back in reverse direction. Thus, by using at least twodisplacers located at different site of the surface area of the firstsurface and/or the second surface that are operated in oppositedirections, as described above, a continuous displacement, and thus alsoa mixing, of a sample within the reaction chamber may be achieved.

In a particularly preferred embodiment of the present invention, thesubsequent reduction and re-increase of the distance between at leastone or more parts of the first surface and the second surface isrepeated at least twice. The extent to which the distance between atleast one or more parts of the first surface and the second surface isreduced during two or more consecutive “compression/relaxation cycles”is preferably be the same. That is, the distance between the twosurfaces in the compressed state of the device remains constant.However, it is also possible to vary the extent to which the distancebetween at least one or more parts of the first surface and the secondsurface is reduced during two or more consecutive“compression/relaxation cycles”, for example by increasing the extentwith an increasing number of cycles. Preferably, this subsequentreduction and re-increase of the distance is repeated until the sampleis evenly distributed within the reaction chamber, i.e. equilibriumconditions have been established, which is essential for the reliabilityof quantitative analyses such as the counting of particles in a sample.Thus, when performing detection within a particular “detection zone” ofthe reaction chamber the determination of the mean value of repeateddetection steps within this area will provide an accurate measure forthe actual number of particles within the entire reaction chamber(provided that the volumes of the entire reaction chamber as well asthat of the “detection zone” are known). The number of cycles ofreduction and re-increase that can be performed is within the opinion ofthe practitioner. Typically, the number of cycles is in a range of 2 to2000, preferably in a range of 10 to 1500, more preferably in a range of50 to 1000 and particularly preferably in a range of 100 to 500.

According to the invention, the detection/determination of a valueindicative for the presence and/or the number of the one or more speciesof the particles may be performed after each cycle of reducing andre-increasing of the distance between the first surface and the secondsurface of the reaction chamber, wherein the detection is preferablyperformed after said distance has been reduced. However, it is alsopossible to repeat the detection several times, for example after everysecond or every fifth reduction/re-increase cycle. Furthermore, it ispossible to perform the detection only once after the completion of thelast reduction/re-increase cycle. In a preferred embodiment, thedetection is performed after each reduction/re-increase cycle. In otherpreferred embodiments of the invention, detection further comprisesdetermining the mean value of the results obtained in the detectionsteps performed by then, as a measure allowing quantitative analysesbeing indicative for the total number of one or more species ofparticles being present in a sample. For example, after the secondreduction/re-increase cycle the mean value of the results obtained afterthe first and the second reduction/re-increase cycle, respectively, iscalculated; after the third reduction/re-increase cycle the mean valueof the results obtained after the first, the second, and the thirdreduction/re-increase cycle, respectively, is calculated, and so forth.The data obtained in one or more cycles of detection may be analyzed andmathematically processed using appropriate computer software known bypersons skilled in the art in order to determine the presence and/or tocalculate the number of particles.

Depending on the particular type of particle(s) to be detected as wellas the nature of detectable markers used detection can be performed byvarious methods, all of them established in the art (see, for example,Ausubel, F. M. et al., supra; Coligan, J. E. et al, (2000) CurrentProtocols in Protein Sciences, Wiley & Sons, Hoboken, N.J.; andLottspeich, F., and Zorbas H., supra).

Thus, performing the method according to the present invention generallydoes not require the use of sophisticated or bulky instrumentation butrather involves simple detection systems, preferably based on themeasurement of parameters such as fluorescence, optical absorption,resonance transfer, and the like. Particularly preferred detectionsystems according to the invention are based on absorption measurementssuch as turbidimetry and nephelometry, as already described above, whichmay be achieved with photometric devices established in the art. Eventhough their simplicity these methods enable accurate determinations notonly of the presence of a one or more given species of particles in asample but also of their number (for review see, for example, Tietz, N.O. (ed.). Textbook of Clinical Chemistry. WB Saunders Co., Philadelphia,Pa., pp. 78-97)

Further particularly preferred detection systems are based “classical”methods for measuring a fluorescent signal such as epifluorescence ordark field fluorescence microscopy (reviewed, e.g., in: Lakowicz, J. R.(1999) Principles of Fluorescence Spectroscopy, 2^(nd) ed., PlenumPublishing Corp., NY).

Further fluorescence detection methods that may also be used in theinvention include inter alia total internal fluorescence microscopy(see, e.g., Axelrod, D. (1999) Surface fluorescence microscopy withevanescent illumination, in: Lacey, A. (ed.) Light Microscopy inBiology, Oxford University Press, New York, 399-423)., fluorescencelifetime imaging microscopy (see, e.g., Dowling, K. ct al. (1999). J.Mod. Optics 46, 199-209), fluorescence resonance energy transfer (see,e.g., Periasamy, A. (2001) J. Biomed. Optics 6, 287-291),bioluminescence resonance energy transfer (see, e.g., Wilson, T., andHastings, J. W. (1998) Annu. Rev. Cell Dev. Biol. 14, 197-230), andfluorescence correlation spectroscopy (see, e.g., Hess, S. T. et al.(2002) Biochemistry 41, 697-705).

Alternatives for the above-mentioned detection systems include whitelight setups, as described, for example, in WO 00/12759, WO 00/25113,and WO 96/27025; confocal systems, as described, for example, in U.S.Pat. No. 5,324,633, U.S. Pat. No. 6,027,880, U.S. Pat. No. 5,585,639,and WO 00/12759; confocal excitation systems based on Nipkow discs, asdescribed, for example, in U.S. Pat. No. 5,760,950; large-scaleintegrated fluorescence detection systems using micro-optics, asdescribed, for example, in WO 99/27140; and laser scanning systems, asdescribed, for example, in WO 00/12759. A general description ofdetection methods using such conventional detection systems can befound, for example, in U.S. Pat. No. 5,324,633.

In addition, as already described above, electrochemical detectionmethods may be used, for example by measuring the alteration of redoxpotentials via electrodes connected to the first surface and/or thesecond surface (see, e.g., Zhu, X. et al. (2004) Lab Chip. 4, 581-587)or by cyclic voltometry (see, e.g., Liu, J. et al. (2005) Anal. Chem.77, 2756-2761; and Wang, J. (2003) Anal. Chem. 75, 3941-3945).Furthermore, an electric detection method can be employed, for exampleby impedance measurement (see, e.g., Radke, S. M. et al. (2005) Biosens.Bioelectron. 20, 1662-1667). Detection may also be accomplished by meansof detecting acoustic surface waves, as described, e.g., in Z.Guttenberg ct al. (2005) Lab Chip. 5, 308-317.

In specific embodiments of the present invention, detection is performedusing FRET or BRET, which are based on the respective formation offluorescence or bioluminescence quencher pairs, so that a fluorescencesignal only occurs, if a target molecule has bound to a capture moleculeimmobilized on the porous matrix. The use of FRET is also described,e.g., in Liu, B. et al. (2005) Proc. Natl. Acad. Sci. USA 102, 589-593;and Szollosi, J. et al. (2002) J. Biotechnol. 82, 251-266. The use ofBRET is detailed, for example, in Prinz, A. et at. (2006) Chembiochem.7, 1007-1012; and Xu, Y. et al. (1999) Proc. Natl. Acad. Sci. USA 96,151-156.

The invention is further described by the following figures andexamples, which are solely for the purpose of illustrating specificembodiments of this invention, and are not to be construed as limitingthe scope of the invention in any way.

DETAILED DESCRIPTION OF THE FIGURES

FIG. 1 depicts a schematic cross-sectional illustration of an assaydevice according to the present invention. The reaction chamber of thedevice is defined by the first surface, the second surface as well asthe lateral sidewalls, wherein at least the central area of the secondsurface is made of an elastically deformable material. The devicefurther comprises a displacer in form of a displacement body locatedopposite to the second surface. The distance between the first surfaceand the second surface is variable by applying pressure to the secondsurface via the displacer. The reaction chamber is located within achamber body and scaled. An optical detection system is located oppositeto first surface of the reaction chamber.

FIG. 2 is a schematic illustration of an inventive assay employing adevice comprising two displacers in form of displacement bodies. One ofthese displacers is located opposite to the first surface, the other oneopposite to the second surface of the device. Furthermore, thedisplacers are positioned in such a way that they are located oppositerelative to each other. The first surface and the second surface aremade of an elastically deformable material at least in the respectivesurface areas located opposite to the displacers. A sample comprisingmultiple particles has been positioned within the reaction chamber alongwith a label having binding affinity for a particular species ofparticles. By applying pressure to the first surface and the secondsurface via the two displacers the distance between said surfacesbecomes reduced in the central part of the reaction chamber thus causingan at least partial displacement of the sample within the reactionchamber (preferably to compensation zones not shown). In addition, byreducing said distance between the two surfaces a “gap” is formed inwhich detection of the specifically labeled particles takes place byrecording a value indicative for their presence and/or number.Afterwards, it is preferred to re-increase the distance between thesurfaces by resetting the two displacers to their original positions(i.e. by discontinuing applying pressure to the surfaces). The displacedsample will thus “move back” and becomes re-distributed throughout thereaction chamber before reducing the distance again. Determining themean value of the results obtained during repeated detection stepsperformed within the “detection zone” will provide an accurate measurefor the total number of the labeled particles to be detected within theentire reaction chamber (provided that the volumes of the reactionchamber as well as that of the “detection zone” are known).

FIG. 3 is a schematic illustration of an alternative inventive assayemploying a device comprising two displacers in form of displacementbodies that are located opposite to the same surface (referred to as the“first surface” below) at the two terminal regions of the device. Thecentral part of the reaction chamber is configured as a capillary gaphaving a defined volume. The respective surface areas of the firstsurface located opposite the two displacers are made of an elasticallydeformable material. A sample comprising multiple particles, has beenpositioned within the reaction chamber along with a label having bindingaffinity for a particular species of particles. By applying pressure tothe first surface via the first displacer at one terminal region of thereaction chamber the distance between the first and the second surfacebecomes reduced in this part of the reaction chamber causing adisplacement of the sample within the reaction chamber towards the otherterminal region. Consequently, the respective second displacer locatedat the other terminal region is pushed by the displaced liquid backwardsin its initial position resulting in an increase of the distance betweenthe first surface and the second surface in this part of the reactionchamber. The displacement of the sample results in the positioning of adistinct subset of the labeled particles present in the reaction chamberwithin the capillary gap where detection takes place. By subsequentlyapplying pressure to the first surface via the second displacer thesample is displaced within the reaction chamber in “backwards direction”resulting in the positioning of another distinct subset of the labeledparticles within the capillary gap. Thus, by using the two displacersthat are operated in opposite directions a continuous displacement, andthus also a mixing of the sample within the reaction chamber isachieved. By determining the mean value of the results obtained duringrepeated detection steps performed within the “detection zone” the totalnumber of the labeled particles to be detected within the entirereaction chamber can be calculated (provided that the volume of thereaction chamber is known as well).

FIG. 4 illustrates a specific embodiment of inventive devices andsystems as well as the corresponding mode of operation. FIG. 4A (toppanel) schematically depicts a singular device or part which isconfigured as a channel defined by a first surface and a second surfaceand running out at one end in a compensation zone or reservoir. At leastpart of the reservoir is made of an elastically deformable material.Within said reservoir reagents (such as buffers or labels) may beprovided, preferably in dry form. A displacer is located opposite to onesurface of the reservoir (not shown). At the opposite end of the devicea sample introduction passage (i.e. a sample loading zone) is provided.The channel encompasses a particular detection area (i.e. a detectionzone). At least two such devices or parts may be assembled to amultipart system comprising separate reaction chambers in order toperform multiple assays of one sample in parallel. The respectivereaction chambers are in communication with each other via a commonsample introduction passage. FIG. 4A (middle and bottom panel) depictstwo exemplary embodiments of such a multipart system comprising two andthree devices, respectively. FIG. 4B schematically illustrates how theunit (either as a single device or as part of a multipart system) isoperated. The upper panel is a cross-sectional view of the device in itsinitial state. The channel encompassing a particular detection area isdelimited by a rigid first surface (hatched) and a second surface madeof an elastically deformable material (“cover membrane”). A displacer(“stencil”) is pressed towards the second surface of the reservoirresulting in a reduction of the distance between the first surface andthe second surface in this surface area, preferably to a value of zeroor almost zero. A drop of a liquid sample is placed in the sampleintroducing passage and introduced into the reaction chamber by means ofnegative pressure resulting from a release of the displacer (i.e. bymoving it in backwards direction), thus causing a relaxation of thereservoir (FIG. 4B, middle panel). Finally, the displacer is againpressed towards the second surface of the reservoir. Thus, by anotherreduction of the distance between the two surfaces in this surface areathe sample becomes at least in part displaced within the reactionchamber. Therefore, by repeating the consecutive compression andrelaxation of the reservoir an agitation/mixing of the sample can beaccomplished before detection of one or more species of particles isperformed (FIG. 4B, lower panel).

FIG. 5 depicts the results of an assay according to the invention fordetermining the number of CD4+ cells in human blood. The CD4+ cells of ahuman blood sample were labeled with an anti-CD4phycoerythrin-conjugated monoclonal antibody using the CD3CD4 kit (GEHealthcare Life Sciences, Heidelberg, Germany) according to theinstructions of the manufacturer. The blood sample was diluted to aconcentration of less than 500 cells/μl and analyzed in a reactionchamber configured as a capillary channel that is made ofpolydimethylsiloxane. The reaction chamber has a total volume ofapproximately 2 μl. After introducing the sample into the reactionchamber by means of capillary forces it was agitated, by applyingpressure to one surface of the reaction chamber via a tappet thuscausing a partial displacement of the sample. Displacing the sample insuch manner was repeated 10 times. The labeled cells were analyzed bymeans of optical detection using a microscope (Akioskop; Carl ZeissGmbH, Jena, Germany) with a 10× objective (image field 1727×1385 μm) andan FI-FITC filter (Zeiss #09). The exposure rime was 2500 ms. Detectionwas repeated 5-7 times. A representative microscope image is shown. Thenumber of CD4+ cells in the sample was determined using an appropriatecomputer software package (Image J. software version 1.71, a publicdomain Java image processing program inspired by NIH Image) bycalculating the mean value of the cell numbers. obtained for theindividual measurements. Since the image field and thus the volume ofthe “detection zone” as well as the volume of the reaction chamber wereknown, the concentration of CD4+ cells/μl sample could. be calculated.

FIG. 6 depicts a comparison of CD4+ cell numbers in human blood asobtained by flow cytometry and an assay according to the invention,respectively. The CD4+ cells of a human blood sample were labeled asdescribed in FIG. 5. Samples were analyzed using a Guava PCA flowcytometer (Guava Technologies, Inc., Hayward, Calif., USA; excitationwavelength: 532 nm, emission wavelength: 580-583 nm) according to themanufacturer's instructions as well as by an inventive assay asdescribed in FIG. 5. The results are mean values±SEM of 5-7 measurementsand are expressed as numbers of CD4+ cells per μl blood.

EXAMPLES Example 1 Determining the Presence of CD4+ Cells in Human Blood

The CD4+ cells of a human blood sample were labeled with anti-CD4phycoerythrin-conjugated monoclonal antibody (CD4-PE) using the CD3CD4kit (GE Healthcare Life Sciences, Heidelberg, Germany) according to theinstructions of the manufacturer. In brief, 10 μl blood were mixed with2 μl CD4-PE (1:25 diluted stock solution) and 2 μl 20 mM EDTA. Then, themixture was incubated for 15 min, in the dark.

The blood sample was diluted to a concentration of less than 500cells/μl (a higher concentration would interfere with proper handling ofthe device) and analyzed in a reaction chamber configured as a capillarychannel that is made of polydimethylsiloxane, i.e. an elasticallydeformable material. The reaction chamber is 2.048 mm×0.04 mm×24 mm insize and thus has a total volume of approximately 2 μl.

For introducing the sample 2.5 μl of the diluted blood sample wereplaced adjacent to the opening of the channel and introduced bycapillary forces without further external manipulation. Afterwards, thereaction chamber was agitated by applying pressure to one surface of thereaction chamber via a tappet (or a forceps) thus causing a partialdisplacement of the sample. Displacing the sample in such manner wasrepeated 10 times.

The labeled cells were analyzed by means of optical detection using amicroscope (Akioskop; Carl Zeiss GmbH, Jena, Germany) with a 10×objective (image field 1727×1385 μm) and an F1-FITC filter (Zeiss #09).The exposure rime was 2500 ms. Detection was repeated 5-7 times. Arepresentative microscope image is shown in FIG. 5.

The number of CD4+ cells in the sample was determined using anappropriate computer software package (Image J software version 1.71, apublic domain Java image processing program inspired by NIH Image) bycalculating the mean value of the cell numbers obtained for theindividual measurements. Since the image field and thus the volume ofthe “detection zone” as well as the volume of the reaction chamber wereknown, the concentration of CD4+ cells/μl sample could be calculated.

Example 2 Comparison of Flow Cytometry and an Inventive Assay forDetermining the Number of CD4+ Cells in Human Blood

In order to test the suitability of the inventive assay for quantitativeanalyses it was compared to conventional flow cytometry for determiningCD4+ cell numbers in human blood. The CD4+ cells of a human blood samplewere labeled as described in Example 1. Samples were analyzed using aGuava PCA flow cytometer (Guava Technologies, Inc., Hayward, Calif.,USA; excitation wavelength: 532 nm, emission wavelength: 580-583 nm)according to the manufacturer's instructions as well as by an inventiveassay as described in Example 1. The results are shown in FIG. 6 andrepresent mean values±SEM of 5-7 measurements. The results are expressedas numbers of CD4+ cells per μl blood.

The mean values obtained for the two methods as well as the respectivedifferences in percent are also summarized in the following table.

Assay no. Mean Flow Mean Assay Δ [%] 1 1234 1114 9.78 2 1208 1330 9.14 31212 1245 2.66 4 1198 1162 3.01 5 1169 1131 3.34 6 1011 710 29.83 7 11181064 4.85 8 1008 968 4.03 9 1058 1170 9.58

As can be seen, in five of the nine independent assays performed thevariation between the two methods is less than 5%, in three cases it isless than 10%. Only in assay no. 6 a higher variation of about 30% hasbeen obtained which may be result from the fact that in this case thetwo analyses have not been performed in parallel, i.e. with identicalblood samples. The other results, however, clearly indicate, thesuitability of the inventive method for the quantitative detection ofparticles in biological samples.

The present invention illustratively described herein may suitably bepracticed in the absence of any element or elements, limitation orlimitations, not specifically disclosed herein. Thus, for example, theterms “comprising”, “including”, “containing”, etc. shall be readexpansively and without limitation. Additionally, the terms andexpressions employed herein have been used as terms of description andnot of limitation, and there is no intention in the use of such termsand expressions of excluding any equivalents of the features shown anddescribed or portions thereof, but it is recognized that variousmodifications are possible within the scope of the invention claimed.Thus, it should be understood that although the present invention hasbeen specifically disclosed by preferred embodiments and optionalfeatures, modification and variation of the inventions embodied thereinherein disclosed may be resorted to by those skilled in the art, andthat such modifications and variations are considered to be within thescope of this invention.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the invention. This includes the genericdescription of the invention with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein.

Other embodiments are within the following claims. In addition, wherefeatures or aspects of the invention are described in terms of Markushgroups, those skilled in the art will recognize that the invention isalso thereby described in terms of any individual member or subgroup ofmembers of the Markush group.

The invention claimed is:
 1. A method for the qualitative and/orquantitative detection of particles, comprising: a) positioning a samplecomprising one or more species of particles to be detected in a reactionchamber comprising a capillary gap defining a volume; b) positioning ofat least a subset of the one or more species of particles to be detectedin the capillary gap by one or more displacers; c) detecting multipleparticles in the capillary gap, and thereby determining a valueindicative of the number of particles belonging to at least one ofspecies of particles.
 2. The method of claim 1, wherein the reactionchamber comprises a first surface and a second surface opposite to thefirst surface and comprising an area made of an elastically deformablematerial, and wherein the positioning of at least a subset of the one ormore species of particles comprises varying the distance between thefirst surface and the second surface in the area made of an elasticallydeformable material.
 3. The method of claim 2, wherein varying thedistance includes applying a pressure to the elastically deformablematerial by the one or more displacers.
 4. The method of claim 2,wherein at least a part of the sample is displaced from the capillarygap by varying the pressure applied to the elastically deformablematerial by the one or more displacers.
 5. The method of claim 4,wherein varying the pressure includes increasing the pressure.
 6. Themethod of claim 5, wherein after displacing at least a part of thesample an increased pressure is subsequently decreased.
 7. The method ofclaim 1, further comprising calculating the number of the particlesbelonging to the at least one of species in the sample from the valueindicative of the number of the particles belonging to the at least oneof species and the volume defined by the capillary gap.
 8. The method ofclaim 1, wherein positioning the sample includes introducing one or moreagents each comprising one or more detectable moieties into the reactionchamber.
 9. The method of claim 8, wherein the one or more agents have abinding affinity for one or more of the particles to be detected. 10.The method of claim 9, wherein the one or more agents and the one ormore species of particles to be detected are allowed to form molecularinteractions with each other, and wherein in step (c) said molecularinteractions are detected.
 11. The method of claim 1, wherein detectingmultiple particles includes optically detecting multiple particles atthe same time.
 12. A method for the qualitative and/or quantitativedetection of particles, comprising: a) positioning a sample comprisingone or more species of particles to be detected in a reaction chambercomprising a capillary gap defining a volume; b) positioning of at leasta subset of the one or more species of particles to be detected in thecapillary gap by one or more displacers; c) imaging a detection zone inthe capillary gap; d) determining from the image a value indicative ofthe number of particles belonging to at least one of species ofparticles.
 13. The method of claim 12, further comprising: e) imaging asecond detection zone in the capillary gap; and f) determining from thesecond image a value indicative of the number of particles belonging toat least one of species of particles.
 14. The method of claim 13,wherein steps d) and f) determine a value indicative of the number ofparticles belonging to the same species of particles.
 15. The method ofclaim 14, further comprising: g) determining a mean value of the resultsobtained in steps d) and f).
 16. The method of claim 15, furthercomprising: h) calculating the total number of particles belonging tothe species of particles in the reaction chamber from the valuesdetermined in steps d), f), g), or a combination thereof.
 17. A methodfor the qualitative and/or quantitative detection of particles,comprising: a) positioning a sample comprising one or more species ofparticles to be detected in a reaction chamber comprising a capillarygap defining a volume; b) positioning of at least a subset of the one ormore species of particles to be detected in the capillary gap by one ormore displacers; c) imaging a detection zone in the capillary gap; d)determining from the image a value indicative of the number of particlesbelonging to at least one of species of particles; e) imaging a seconddetection zone in the capillary gap; f) determining from the secondimage a value indicative of the number of particles belonging to atleast one of species of particles; and g) calculating the total numberof particles belonging to the species of particles in the reactionchamber from the values determined in steps d), f), or a combinationthereof.